The induction of specific transplantation tolerance, which might eliminate both the need for long-term immunosuppressive medications and the persistent problem of chronic rejection, remains a major goal of clinical organ transplantation. Previous studies from our laboratory have demonstrated that the establishment of mixed lymphohematopoietic chimerism provides an effective means for the induction of long-term transplantation tolerance across major histocompatibility barriers in mice. Based on these murine studies, we successfully developed a non-myeloablative preparative regimen that permits the induction of mixed chimerism and renal allograft tolerance following bone marrow transplantation in MHC-mismatched cynomolgus monkeys, without GvHD. However, to apply this regimen to a clinical setting, several modifications will be necessary. Therefore, the major goals of this proposal are to achieve consistent tolerance, to reduce further the morbidity of the preparative regimen, and to extend its use to the transplantation of cadaveric organs. Specifically, we will: 1) evaluate the addition of costimulatory blockade (with anti-CD40L monoclonal antibody and/or CTLA4-Ig) to the preparative regimen; 2) attempt to minimize the radiation requirements of the preparative regimen by utilizing high-dose mobilized peripheral blood stem cells (PBSC) for the induction of mixed chimerism; and 3) modify the preparative regimen to improve its applicability to cadaver-donor transplantation in humans. In addition, we will investigate and compare the underlying mechanism of tolerance induced by each of these non-myeloablative regimens, with particular emphasis on distinguishing central from peripheral mechanisms. These analyses should provide valuable information for extending this approach to clinical allotransplantation.